This application note was produced in collaboration with Dylan D. Doxsey and Kuang Shen at University of Massachusetts Chan Medical School.
In this application note, we demonstrate how single-molecule Förster Resonance Energy Transfer (smFRET) can be performed using the EI-FLEX to resolve the conformational changes of a highly dynamic heterodimeric protein, Rag GTPase, that binds nucleotides and the serine/threonine protein kinase complex mTORC1. smFRET measurements were acquired across a range of conditions to discern how the global structure of Rag GTPase changes and drives downstream signalling in response to cellular amino acid sensing1.
Overview of this application note:
- Rag GTPase binding of guanine diphosphate (GDP) and or guanine triphosphate (GTP) drives global conformational changes that differ depending on the combination of bound nucleotides
- Dual binding of GTP induces inter-subunit cross-talk that stabilises Rag GTPase in order for hydrolysis to occur
- Mutations in the nucleotide-binding domain that hyperactivate or inhibit mTORC signalling can be linked to Rag GTPase structural changes, independent of correct nucleotide binding
- Binding of the mTORC subunit Raptor causes Rag GTPase to adopt an open, bound conformation
Figure 1 – smFRET detects dynamic conformational changes of Rag GTPase in response to nucleotide binding
Modified figure from publication by Doxsey and Shen1 .
